Please help as I am going to start it for the first time in my lab!!
Hello, it would help if you explained more about what is the purpose for what you need FACS. There are several considerations before starting, like the type of sample (cells vs tissue) and the population of study (markers-based, cell cycle in general, etc).
This is a link for the protocol we use in our lab. It is the simplest FACS method we use and is only to determine cell cycle distribution in our samples.
http://cytometry.arl.arizona.edu/index.php/resources/20-generic-category/37-cell-cycle.html
To sort based on specific markers, you can use antibodies tagged with different fluorophores.
Good Luck!!
@ Alejandro Martinez Chibly: I have to see the effect of my drug on cancer cell lines, I have already performed DNA fragmentation.
You need to define what you are looking for!
What is the nature of the cellular effects from your drug? Physical changes in cell structure? Protein markers overexpressed? Diminishing? Expressed de-novo? Once you define what you are looking for you can obtain antibodies and check it out.
Physical cellular changes are easy to observe and require no staining of the cells.
You can simply follow the changes in the forward and side scatter parameters.
Dying or dead cells should shift down in both parameters.
You could (should) stain dead cells for viability tests, using PI,7AAD etc.
For protein markers you would have to buy the appropriate fluorescent antibodies.
Protocols are basic: suspend your cells and transfer them into FACS buffer. Incubate with antibodies or dyes (according to commercial instructions), centrifuge cells and wash few times by suspending in FACS buffer. Finally re-suspend cell pellet in PBS before reading.
Depending on your FACS machine you can run from 4 (but usualy 3) antibodies together in one vial staining 4 separate markers as in the BD FACScallibur. Up to 17 (if I remember correctly) in the more advanced BD LSR III
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